Receptor-mediated-endocytosis (RME) is a system for the movement into the cell of macromolecules. One of the best characterized systems utilizing RME is the hepatic receptor for asialoglycoproteins (ASGP-R). At present there is no complete picture of the structure, function, intracellular movement and modulation of any one particular cell surface receptor. The ASGP-R is well-suited for these studies since I have purified receptor, anti-receptor antibodies, purified ligands, anti-ligand antibodies and a continuous human hepatoma cell line which expresses abundant ASGP-R. In order to examine the movement and function of the ASGP-R during biosynthesis and endocytosis, I shall identify and characterize the human ASGP-R and generate additional high-affinity antibodies to be used as probes in later experiments. Receptor will be further identified on the hepatoma cell via surface labelling and with a novel photoaffinity reagent. The kinetics of biosynthesis, modification and intracellular movement of newly synthesized ASGP-R will be examined in these cells using pulse/chase labelling with 35S-amino acid and immunoprecipitation. The products, modification and kinetics of cell-free synthesis of the human ASGP-R will be performed from isolated mRNA following immunoselection with anti-receptor antibody. The intracellular movement of the ASGP-R during RME will be addressed biochemically via use of direct labelling of the receptor molecule with photoaffinity label as well as antibody. Constitutive recycling of the receptor will be elucidated using antibody labelling and drugs which interfere with receptor movement. In addition, direct morphological evaluation of ASGP-R localization, movement during RME and localization of the site of drug effects will be accomplished with colloidal gold immunocytoelectron microscopy. Cloning of the cDNA for the human ASGP-R from enriched mRNA will provide probes for future studies of receptor structure and gene expression in physiological and pathophysiological states.